scholarly journals Augmentation of Gene Expression and Production of Promatrix Metalloproteinase 2 by Propionibacterium acnes-Derived Factors in Hamster Sebocytes and Dermal Fibroblasts: A Possible Mechanism for Acne Scarring

2011 ◽  
Vol 34 (2) ◽  
pp. 295-299 ◽  
Author(s):  
Takashi Sato ◽  
Hirokazu Kurihara ◽  
Noriko Akimoto ◽  
Norihisa Noguchi ◽  
Masanori Sasatsu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Propionibacterium Acnes ◽  
Dermal Fibroblasts ◽  
Acne Scarring

scholarly journals Dopamine adjusts the circadian gene expression of Per2 and Per3 in human dermal fibroblasts from ADHD patients

2021 ◽  
Author(s):  
Frank Faltraco ◽  
Denise Palm ◽  
Adriana Uzoni ◽  
Lena Borchert ◽  
Frederick Simon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dermal Fibroblast ◽  
Sleep Onset ◽  
Dermal Fibroblasts ◽  
Control Group ◽  
Adhd Group ◽  
Healthy Controls ◽  
Circadian Gene ◽  
Significant Difference ◽  
Circadian Preference

AbstractA link between dopamine levels, circadian gene expression, and attention deficit hyperactivity disorder (ADHD) has already been demonstrated. The aim of this study was to investigate the extent of these relationships by measuring circadian gene expression in primary human-derived dermal fibroblast cultures (HDF) after dopamine exposure. We analyzed circadian preference, behavioral circadian and sleep parameters as well as the circadian gene expression in a cohort of healthy controls and participants with ADHD. Circadian preference was evaluated with German Morningness-Eveningness-Questionnaire (D-MEQ) and rhythms of sleep/wake behavior were assessed via actigraphy. After ex vivo exposure to different dopamine concentrations in human dermal fibroblast (HDF) cultures, the rhythmicity of circadian gene expression (Clock, Bmal1, Per1-3, Cry1) was analyzed via qRT-PCR. We found no statistical significant effect in the actigraphy of both groups (healthy controls, ADHD group) for mid-sleep on weekend days, mid-sleep on weekdays, social jetlag, wake after sleep onset, and total number of wake bouts. D-MEQ scores indicated that healthy controls had no evening preference, whereas subjects with ADHD displayed both definitive and moderate evening preferences. Dopamine has no effect on Per3 expression in healthy controls, but produces a significant difference in the ADHD group at ZT24 and ZT28. In the ADHD group, incubation with dopamine, either 1 µM or 10 µM, resulted in an adjustment of Per3 expression to control levels. A similar effect also was found in the expression of Per2. Statistical significant differences in the expression of Per2 (ZT4) in the control group compared to the ADHD group were found, following incubation with dopamine. The present study illustrates that dopamine impacts on circadian function. The results lead to the suggestion that dopamine may improve the sleep quality as well as ADHD symptoms by adjustment of the circadian gene expression, especially for Per2 and Per3.

scholarly journals OP0248 DEVELOPMENT OF A 3D SKIN MICROTISSUE MODEL FOR FIBROTIC DISEASES

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 151.2-152
Author(s):  
E. Pachera ◽  
G. Kania ◽  
A. Juengel ◽  
M. Calcagni ◽  
O. Distler
Keyword(s):  
Gene Expression ◽  
Noncoding Rna ◽  
Clinical Investigation ◽  
3D Culture ◽  
Human Keratinocytes ◽  
Dermal Fibroblasts ◽  
Proof Of Concept ◽  
Research Support ◽  
Preclinical Research ◽  
Matrix Gene

Background:Traditional preclinical approaches, such as two-dimensional cell culture and animal models, are often inadequate to mimic the pathophysiological features of complex diseases such as systemic sclerosis (SSc). Human specific targets, such as the recently described pro-fibrotic long non coding RNA (lncRNA) H19X1, are becoming increasingly relevant in preclinical research, creating the need of new strategies and tools in translational medicine. The employment of novel three-dimensional (3D) culture systems, where multiple cell types are included, is filling an important gap left by the traditional preclinical methods.Objectives:To develop an easy to produce 3D fibrotic skin microtissues model for translational proof of concept studies.Methods:Two thousand five hundred dermal fibroblasts isolated from skin of SSc patients were seeded in ultra-low attachment 96-well plates. Fibroblast were let to aggregate into spheres for 48h. Two thousand five hundred primary normal human keratinocytes were added to the culture and let to layer onto the fibroblast spheres for 72h. H19X silencing experiments were used as proof of concept studies. H19X silencing with antisense oligonucleotides or transfections with a scrambled control were performed in fibroblasts prior to the sphere formation for 24h. TGFβ (10 ng/ml) was added to microtissue to exacerbate the fibrotic phenotype. Haematoxylin eosin staining as well as immunohistochemistry staining for vimentin and cytokeratin 10 was performed. Skin microtissues were processed for RNA and protein isolation. Pro-collagen Iα1 and fibronectin were quantified in the supernatants with ELISA.Results:The microtissues presented a core of SSc fibroblast as revealed by vimentin staining and an external layer of keratinocytes as revealed by cytokeratin 10 staining, mimicking the human skin architecture. Gene expression analysis following TGFβ stimulation displayed induced expression of extracellular matrix gene COL1A1 (p=0.044) and the myofibroblast marker ACTA2 (p=0.018), indicating that the microtissues were able to develop a fibrotic response. Microtissues, where H19X was silenced, displayed reduced gene expression of COL1A1 and ACTA2 after TGFβ stimulation (COL1A1 p=0.007, ACTA2 p=0.045). Additionally, H19X silencing led to lower levels of αSMA protein expression (p=0.009) and pro-collagen1α1 secretion (p=0.039) in the supernatant of the microtissue cultures as revealed by Western Blot and ELISA, respectively. FN1 expression and fibronectin protein levels were not significantly reduced in the microtissues after H19X silencing.Conclusion:We were able to produce a 3D microtissue resembling skin architecture that can respond to fibrotic stimuli. Knockdown experiments of pro-fibrotic lncRNA H19X confirmed the potential of the model as screening platform for novel pro-fibrotic effectors. A future aim will be to optimize the model for high-throughput automated screening platforms.References:[1]Pachera, E., et al. (2020). “Long noncoding RNA H19X is a key mediator of TGF-β–driven fibrosis.” The Journal of Clinical Investigation 130(9): 4888-4905.Disclosure of Interests:Elena Pachera: None declared, Gabriela Kania: None declared, Astrid Juengel: None declared, Maurizio Calcagni Speakers bureau: Arthrex, Consultant of: Medartis, Arthrex, SilkBiomaterials, Grant/research support from: Medartis, Oliver Distler Speakers bureau: Actelion, Bayer, Boehringer Ingelheim, Medscape, Novartis, Roche, Consultant of: Abbvie, Actelion, Acceleron Pharma, Amgen, AnaMar, Arxx Therapeutics, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, ChemomAb, Corpuspharma, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, -Kymera, Medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi, UCB, Grant/research support from: Abbvie, Actelion, Bayer, Boehringer Ingelheim, Kymera Therapeutics, Mitsubishi Tanabe

scholarly journals Wound Healing Promotion by Hyaluronic Acid: Effect of Molecular Weight on Gene Expression and In Vivo Wound Closure

2021 ◽  
Vol 14 (4) ◽  
pp. 301
Author(s):  
Yayoi Kawano ◽  
Viorica Patrulea ◽  
Emmanuelle Sublet ◽  
Gerrit Borchard ◽  
Takuya Iyoda ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Weight ◽  
Wound Healing ◽  
Hyaluronic Acid ◽  
Healing Process ◽  
Dermal Fibroblasts ◽  
Water Soluble ◽  
Wound Healing Process ◽  
And Migration ◽  
Proliferation And Migration

Hyaluronic acid (HA) has been known to play an important role in wound healing process. However, the effect of molecular weight (MW) of exogenously administered HA on the wound healing process has not been fully understood. In this study, we investigated HA with different MWs on wound healing process using human epidermal keratinocytes and dermal fibroblasts. Cell proliferation and migration ability were assessed by water soluble tetrazolium (WST) assay and wound scratch assay. We examined the effect of HA addition in a full-thickness wound model in mice and the gene expression related to wound healing. Proliferation and migration of HaCaT cells increased with the increase of MW and concentration of HA. Interleukin (IL-1β), IL-8 and vascular endothelial growth factor (VEGF) as well as matrix metalloproteinase (MMP)-9 and MMP-13 were significantly upregulated by high molecular weight (HMW) HA in keratinocytes. Together with VEGF upregulation and the observed promotion of HaCaT migration, HA with the MW of 2290 kDa may hold potential to improve re-epithelialization, a critical obstacle to heal chronic wounds.

scholarly journals SAT0299 PROLIFERATION, MIGRATION AND CONTRACTION ARE DIFFERENT BETWEEN TGFΒ AND PDGF STIMULATED DERMAL FIBROBLASTS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1094.2-1095
Author(s):  
A. S. Siebuhr ◽  
S. F. Madsen ◽  
M. Karsdal ◽  
A. C. Bay-Jensen ◽  
P. Juhl
Keyword(s):  
Gene Expression ◽  
Treatment Initiation ◽  
Human Fibroblasts ◽  
Calf Serum ◽  
Collagen Type I ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Full Time ◽  
Ecm Remodeling ◽  
Full Time Employee

Background:Systemic sclerosis has vascular, inflammatory and fibrotic components, which may be associated with different growth factors and cytokines. Platelet derived growth factor (PDGF) is associated with the vasculature, whereas tumor necrosis factor beta (TGFβ) is associated with inflammation and fibrosis. We have developed a fibroblast model system of dermal fibrosis for anti-fibrotic drugs testing, but the effect of the fibroblasts mechanistic properties are unknown.Objectives:We investigated different mechanical capacities of PDGF and TGFβ treated human healthy dermal fibroblasts in the SiaJ setting.Methods:Primary human healthy dermal fibroblasts were grown in DMEM medium containing 0.4% fetal calf serum, ficoll (to produce a crowded environment) and ascorbic acid for up to 17 days. A wound was induced by scratching the cells at 0, 1, 3 or 7 days after treatment initiation. Wound closure was followed for 3 days. Contraction capacity was tested by creating gels of human fibroblasts produced collagens containing dermal fibroblasts and contraction was assessed at day 2 by calculating the percentage of gel size to total well size. Collagen type I, III and VI formation (PRO-C1, PRO-C3 and PRO-C6) and fibronectin (FBN-C) were evaluated by validated ELISAs (Nordic Bioscience). Gene expression was analyzed after 2 days in culture. Statistical analyses included One-way ANOVA and student’s t-test.Results:Generally, PDGF closed the wound in half the time of w/o and TGFβ, when treatment and cells are added concurrently or scratched one day after treatment initiation. When treatments were added 3 or 7 days prior to scratch, the cells treated with PDGF had proliferated to a higher degree than w/o and TGFβ. A consequence of this, was that when cells were scratch the sheet of cells produced was lifted from the bottom and fold over itself, leaving a much greater scratch than in the other treatments. However, despite this increased gap the PDGF treated cells closed the wound at the same time as w/o and TGFβ, confirming the results of the cells scratched at day 0 and 1.Inhibition of contraction by ML-7 of otherwise untreated cells inhibited contraction significantly compared to untreated cells alone (p=0.0009). Contraction was increased in both TGFβ and PDGF treated cells compared to untreated cells (both p<0.0001). TGFβ+ ML-7 inhibited the contraction to the level of w/o (p=0.0024), which was only 35% of ML-7 alone. In the contraction study the cells were terminated after 2 days of culture, thus prior to when biomarker of ECM remodeling is usually assessed. However, FBN-C was detectable and a significant release of fibronectin by TGFβ and PDGF compared to w/o was found in the supernatant (both p<0.0001). The gene expression of FBN was only increased with TGFβ (p<0.05) and not PDGF. ML-7 alone tended to decrease FBN-C and in combination with TGFβ the FBN level was significantly decreased compared to TGFβ alone (p<0.0001). However, the level of TGFβ+ML-7 was significantly higher than ML-7 alone (p=0.038).TGFβ increased the gene expression of most genes assessed, except Col6a1. PDGF increased the gene expression of Col3a1, Col5a1 and Col6a1 above the critical fold change of 2, but only significantly in Col5a1 and Col6a1 (both p<0.05).Conclusion:This study demonstrates that TGFβ and PDGF have different mechanical capacities in human healthy dermal fibroblasts; TGFβ increased gene expression of ECM related genes, such as collagens and have increased FBN release in the supernatant already 2 days after initial treatment. PDGF has increased contraction, proliferation and migratory capacities and less expression of ECM related genes and proteins.Disclosure of Interests:Anne Sofie Siebuhr Employee of: Nordic Bioscience, Sofie Falkenløve Madsen: None declared, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S., Pernille Juhl Employee of: Nordic Bioscience

scholarly journals SAT0298 IS INTERLEUKIN 6 A FACTOR OF FIBROGENESIS IN DERMAL FIBROBLASTS?

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1094.1-1094
Author(s):  
A. S. Siebuhr ◽  
P. Juhl ◽  
M. Karsdal ◽  
A. C. Bay-Jensen
Keyword(s):  
Gene Expression ◽  
Type I Collagen ◽  
Collagen Type ◽  
Collagen Type I ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Dependent Manner ◽  
Full Time ◽  
Collagen Formation ◽  
Type Iii

Background:Interleukin 6 (IL-6) is known to have both pro- and anti-inflammatory properties, depending on the receptor activation. The classical IL-6 signaling via the membrane bound receptor is mainly anti-inflammatory, whereas signaling through the soluble receptor (sIL-6R) is pro-inflammatory/pro-fibrotic. However, the direct fibrotic effect of IL-6 stimulation on dermal fibroblasts is unknown.Objectives:We investigated the fibrotic effect of IL-6 + sIL-6R in a dermal fibroblast model and assessed fibrosis by neo-epitope biomarkers of extracellular matrix proteins.Methods:Primary healthy human dermal fibroblasts were grown for up to 17 days in DMEM medium with 0.4% fetal calf serum, ficoll (to produce a crowded environment) and ascorbic acid. IL-6 [1-90 nM]+sIL-6R [0.1-9 nM] alone or in combination with TGFβ [1 nM] were tested in three different donors. TGFβ [1 nM], PDGF-AB [3 nM] and non-stimulated cells (w/o) were used as controls. Tocilizumab (TCZ) with TGFβ + IL-6 + sIL-6R stimulation was tested in one donor. Collagen type I, III and VI formation (PRO-C1, PRO-C3 and PRO-C6) and fibronectin (FBN-C) were evaluated by validated ELISAs (Nordic Bioscience). Western blot analysis investigated signal cascades. Gene expression of selected ECM proteins was analyzed. Statistical analyses included One-way and 2-way ANOVA and area under the curve analysis.Results:formation by the end of the culture period. The fibronectin and collagen type VI signal were consistent between the three tested donors, whereas the formation of type III collagen was only increased in one donor, but in several trials. Type I collagen formation was unchanged by IL-6 + sIL-6R stimulation. The gene expression of type I collagen was induced by IL-6 + sIL-6R. Western blot analysis validated trans-signaling by the IL-6+sIL-6R stimulation as expected.IL-6 + sIL-6R stimulation in combination with TGFβ decreased fibronectin levels compared to TGFβ alone but did not reach the level of unstimulated fibroblasts. The formation of collagen type IV was generally unchanged with IL-6 + sIL-6R + TGFβ compared to TGFβ alone. Collagen type I and III formation was more scattered in the signals when IL-6 + sIL-6R was in combination with TGFβ, as the biomarker level could be either decreased or increased compared to TGFβ alone. In two studies the type I collagen level was synergistic increased by IL-6 + sIL-6R + TGFβ, whereas another study found the level to be decreased compared to TGFβ alone. The gene expression of fibronectin and type I collagen was increased with TGFβ +IL-6+sIL-6R compared to TGFβ alone.Inhibition of IL-6R by TCZ in combination with IL-6 + sIL-6R did only decrease the fibronectin level with the lowest TCZ concentration (p=0.03). TCZ alone decreased the fibronectin level in a dose-dependent manner (One-way ANOVA p=0.0002).Conclusion:We investigated the fibrotic response of dermal fibroblasts to IL-6 + sIL-6R stimulation. IL-6 modulated the fibronectin level and modulated the collagen type III formation level in a somewhat dose-dependent manner. In combination with TGFβ, IL-6 decreased collagen type I and IV formation and fibronectin. However, in this study inhibition of IL-6R by TCZ did not change the fibrotic response of the dermal fibroblasts. This study indicated that IL-6 did not induce collagen formation in dermal fibroblasts, except type III collagen formation with high IL-6 concentration.Figure:Disclosure of Interests:Anne Sofie Siebuhr Employee of: Nordic Bioscience, Pernille Juhl Employee of: Nordic Bioscience, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S.

Abstract 16564: Analysis of the Mechanisms of Intravenous Immunoglobulin-Resistant Kawasaki Disease Using iPS Cell Technology

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Kazuyuki Ikeda ◽  
Tomonaga Ameku ◽  
Yui Nomiya ◽  
Masahiro Nakamura ◽  
Satoshi Matsui ◽  
...  
Keyword(s):  
Gene Expression ◽  
Kawasaki Disease ◽  
Intravenous Immunoglobulin ◽  
Expression Profiles ◽  
Systemic Vasculitis ◽  
Dermal Fibroblasts ◽  
Gene Set Enrichment Analysis ◽  
Recurrent Fever ◽  
Ips Cell ◽  
Ivig Resistance

Introduction: Kawasaki disease (KD) is a systemic vasculitis of unknown origin. Although the treatment of intravenous immunoglobulin (IVIG) significantly resolves inflammation, 10-20% of KD patients have persistent or recurrent fever after the administration of IVIG, and IVIG-resistant patients have a particularly high risk of developing coronary artery abnormalities. Hypothesis: The mechanisms of IVIG-resistant KD have been analyzed using the patients’ leukocyte samples. However, vascular endothelial cells (ECs), closely related to the vasculitis of KD, have not been examined in the previous reports. We propose a hypothesis that ECs are mainly involved in the etiology of IVIG-resistance. Methods: The purpose of this study is to establish new in vitro disease models of vasculitis using induced pluripotent stem cell (iPSC) technology, and clarify the mechanisms of IVIG-resistance in KD. Dermal fibroblasts or T cells from 2 IVIG-resistant and 2 IVIG-responsive KD patients were reprogrammed by episomal vectors encoding Oct3/4, Sox2, Klf4, L-Myc, LIN28, and p53 shRNA. The iPSC lines were then differentiated into ECs by using a previously-reported differentiation method, and the EC samples were subjected to the microarray analyses. Results: The KD patient-derived iPSCs could be differentiated into ECs. The gene expression profiles were compared between iPS-derived ECs (iPS-ECs) generated from IVIG-resistant and IVIG-responsive KD patients. We found that 107 genes were at least two fold up-regulated and 101 genes were at least two fold down-regulated in iPS-ECs from IVIG-resistant KD patients compared with those from IVIG-responsive patients. The Principle Component Analysis (PCA) was performed, but the gene expression levels showed no significant differences between the groups. The Gene Set Enrichment Analysis (GSEA) revealed that the gene sets related to IL-6, NRAS (a member of the RAS oncogene family) and breast cancer were up-regulated in iPS-ECs from IVIG-resistant KD patients. Conclusions: Taking into account that the concentration of IL-6 has been reported to be elevated in acute phase of IVIG-resistant KD, our results suggest that the up-regulation of IL-6 related genes in ECs might be involved in the pathogenesis of IVIG-resistant KD.

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